Compositions and methods for determining whether a subject would benefit from co-receptor inhibitor therapy

ABSTRACT

The present invention provides methods and compositions for determining whether a subject would benefit from co-receptor inhibitor therapy. In certain aspects, the methods can be used to determine whether a subject infected with a dual-mixed tropic population of HIV would benefit from CCCR5-inhibitor therapy or CXCR4-inhibitor therapy, the methods comprising determining whether the HIV population is a homogeneous or heterogeneous population of HIV, wherein the nature of the homogenous or heterogenous population of HIV indicates whether the patient would benefit from co-receptor inhibitor therapy.

1. CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 12/527,863, filed Mar. 11, 2010, which is a U.S. National Stageapplication of International Application No. PCT/US2008/002497, filed 26Feb. 2008, which claims the benefit of U.S. Patent Application No.60/903,655, filed 26 Feb. 2007, the contents of which are incorporatedherein by reference in their entirety.

Throughout this application, various publications are referenced byauthor and date within the text. Full citations for these publicationsmay be found listed alphabetically at the end of the specificationimmediately preceding the claims. All patents, patent applications andpublications cited herein are hereby incorporated by reference in theirentirety.

2. BACKGROUND

Enveloped animal viruses attach to and enter the host cell via theinteraction of viral proteins in the virion membrane (envelope proteins)and cell surface proteins (virus receptors). Receptor recognition andbinding are mediated by the surface envelope protein. Virus entry is anattractive target for anti-viral treatment; numerous entry inhibitordrugs that are designed to block HIV receptor (CD4) attachment,co-receptor (CCR5, CXCR4) engagement, or host cell-virus membrane fusionhave been or are currently being evaluated in preclinical or clinicalstudies (Richman, 1998; PhRMA, 1999; Stephenson, 1999). For example, theco-receptor antagonist vicriviroc (SCH-D, Schering Plough), which blocksthe interaction between the viral membrane surface protein (gp120) andCCR5, is currently being evaluated in clinical studies for itseffectiveness as an anti-viral treatment (Shurman, 2004). Other entryinhibitors currently or previously under investigation include UK-427857(maraviroc, Pfizer), TNX-355 (Tanox Inc.), AMD-11070 (AnorMED), Pro 140and Pro 584 (Progenies), FP-21399 (EMD Lexigen), BMS-488043(Bristol-Myers Squibb), INCB9471 (Incyte), KRH-3955 and KRH-314(Kureha), HGSImAb004 (Human Genome Sciences), TRI-999, (Trimeris) andGSK-873,140 (aplaviroc, GlaxoSmithKline). One entry inhibitor, FUZEON®(enfuvirtide; Roche/Trimeris), has been approved for treatment of HIVinfection by the United States Food and Drug Administration.

As these drugs continue to be developed and enter the clinic, assays areneeded that can rapidly and easily identify patient populations that mayclinically benefit from administration of these drugs. These and otherunmet needs are provided by the present invention.

3. SUMMARY

In a first aspect, the invention provides a method for determiningwhether a subject with a dual-mixed tropic population of humanimmunodeficiency viruses (HIV) would benefit from CXCR4-inhibitor orCCR5-inhibitor therapy, comprising: a) determining whether thepopulation is a homogeneous or heterogeneous population; wherein if thepopulation is a homogeneous population, step b of the method isperformed, and wherein if the population is a heterogeneous population,step c of the method is performed; b) determining whether thehomogeneous population is Dual_R tropic or Dual_X tropic or neither(Dual tropic), wherein if the homogeneous population is composed ofDual_R tropic HIV, the subject would benefit from CCR5-inhibitortherapy, wherein if the homogeneous population is composed of Dual_XHIV, the subject would benefit from CXCR4-inhibitor therapy, and whereinif the homogeneous population is neither Dual_R tropic nor Dual_X, thesubject would not benefit from either CCR5-inhibitor or CXCR4-inhibitortherapy; and c) determining the relative proportions of CCR5-tropic,CXCR4-tropic, Dual_R-tropic, Dual_X-tropic, or Dual tropic HIV of theheterogeneous population, wherein

if the heterogeneous population contains i) CCR5-tropic and/orDual_R-tropic, and ii) CXCR4-tropic and/or Dual_X-tropic HIV andcomprises substantially more CCR5-tropic and/or Dual_R-tropic HIV thanCXCR4-tropic and/or Dual_X-tropic HIV, the subject would benefit fromCCR5-inhibitor therapy;

if the heterogeneous population contains i) CCR5-tropic and/or Dual_Rtropic, and ii) CXCR4-tropic and/or Dual_X tropic HIV and comprisescomparable amounts of CCR5-tropic and/or Dual_R tropic and CXCR4-tropicand/or Dual_X tropic HIV, the subject would not benefit from eitherCCR5-inhibitor or CXCR4-inhibitor therapy;

if the heterogeneous population contains 1) CCR5-tropic and/or Dual_Rtropic, and ii) CXCR4-tropic and/or Dual_X tropic HIV and comprisessubstantially more CXCR4-tropic and/or Dual_X tropic HIV thanCCR5-tropic and/or Dual_R tropic HIV, the subject would benefit fromCXCR4-inhibitor therapy;

if the heterogeneous population contains i) CCR5-tropic and/or Dual_Rtropic, and ii) dual-tropic HIV and comprises substantially moreCCR5-tropic and/or Dual_R tropic HIV than dual-tropic HIV, the subjectwould benefit from CCR5-inhibitor therapy;

if the heterogeneous population contains i) CCR5-tropic and/or Dual_Rtropic, and ii) dual-tropic HIV and comprises relatively comparableamounts of CCR5-tropic and/or Dual_R tropic HIV and dual-tropic HIV orcomprises substantially more dual-tropic HIV than CCR5-tropic and/orDual_R tropic HIV, the subject would not benefit from CCR5-inhibitor orCXCR4-inhibitor therapy;

if the heterogeneous population contains i) CXCR4-tropic and/or Dual_Xtropic, and ii) dual-tropic HIV and comprises substantially moreCXCR4-tropic and/or Dual_X tropic HIV than dual-tropic HIV, the subjectwould benefit from CXCR4-inhibitor therapy; if the heterogeneouspopulation contains i) CXCR4-tropic and/or Dual_X tropic, and ii)dual-tropic HIV and comprises relatively comparable amounts ofCXCR4-tropic and/or Dual_X tropic HIV and dual-tropic HIV or comprisessubstantially more dual-tropic HIV than CXCR4-tropic and/or Dual_Xtropic HIV, the subject would not benefit from either CXCR4-inhibitor orCCR5-inhibitor therapy;

if the heterogeneous population contains i) CCR5-tropic and/or Dual_Rtropic, ii) CXCR4-tropic and/or Dual_X tropic, and iii) dual-tropic HIVand comprises substantially more CCR5-tropic and/or Dual_R tropic HIVthan CXCR4-tropic and/or Dual_X tropic and dual-tropic HIV, the subjectwould benefit from CCR5-inhibitor therapy;

if the heterogeneous population contains i) CCR5-tropic and/or Dual_Rtropic, ii) CXCR4-tropic and/or Dual_X tropic, and iii) dual-tropic HIVand comprises relatively comparable amounts of CCR5-tropic and/or Dual_Rtropic HIV and CXCR4-tropic and/or Dual_X tropic and dual-tropic HIV,the subject would not benefit from either CCR5-inhibitor orCXCR4-inhibitor therapy; and

if the heterogeneous population contains i) CCR5-tropic and/or Dual_Rtropic, ii) CXCR4-tropic and/or Dual_X tropic, and iii) dual-tropic HIVand comprises substantially more CXCR4-tropic and/or Dual_X tropic HIVthan CCR5-tropic and/or Dual_R tropic and dual-tropic HIV, the subjectwould benefit from CXCR4-inhibitor therapy.

In certain embodiments, the population is a homogeneous population ofdual tropic viruses. In certain embodiments, the homogeneous populationis Dual_R-tropic. In certain embodiments, the homogeneous population isDual_X-tropic. In certain embodiments, the homogeneous population isneither Dual_R-tropic nor Dual_X-tropic.

In certain embodiments, the population is a heterogeneous population ofmixed virus tropisms. In certain embodiments, the heterogeneouspopulation contains CCR5-tropic and/or Dual_R tropic and CXCR4-tropicand/or Dual_X tropic HIV. In certain embodiments, the populationcomprises substantially more CCR5-tropic and/or Dual_R tropic HIV thanCXCR4-tropic and/or Dual_X tropic HIV. In certain embodiments, thepopulation comprises comparable amounts of CCR5-tropic and/or Dual_Rtropic and CXCR4-tropic and/or Dual_X tropic HIV. In certainembodiments, the population comprises substantially more CXCR4-tropicand/or Dual_X tropic HIV than CCR5-tropic and/or Dual_R tropic HIV.

In certain embodiments, the heterogeneous population containsCCR5-tropic and/or Dual_R tropic and dual-tropic HIV. In certainembodiments, the population comprises substantially more CCR5-tropicand/or Dual_R tropic HIV than dual-tropic HIV. In certain embodiments,the population comprises relatively comparable amounts of CCR5-tropicand/or Dual_R tropic HIV and dual-tropic HIV. In certain embodiments,the population comprises substantially more dual-tropic HIV thanCCR5-tropic and/or Dual_R tropic HIV.

In certain embodiments, the heterogeneous population containsCXCR4-tropic and/or Dual_X tropic and dual-tropic HIV. In certainembodiments, the population comprises substantially more CXCR4-tropicand/or Dual_X tropic HIV than dual-tropic HIV. In certain embodiments,the population comprises relatively comparable amounts of CXCR4-tropicand/or Dual_X tropic HIV and dual-tropic HIV. In certain embodiments,the population comprises substantially more dual-tropic HIV thanCXCR4-tropic and/or Dual_X tropic HIV.

In certain embodiments, the heterogeneous population containsCCR5-tropic and/or Dual_R tropic, CXCR4-tropic and/or Dual_X tropic, anddual-tropic HIV. In certain embodiments, the population comprisessubstantially more CCR5-tropic and/or Dual_R tropic HIV thanCXCR4-tropic and/or Dual_X tropic and dual-tropic HIV. In certainembodiments, the population comprises relatively comparable amounts ofCCR5-tropic and/or Dual_R tropic HIV and CXCR4-tropic and/or Dual_Xtropic and dual-tropic HIV. In certain embodiments, the populationcomprises substantially more CXCR4-tropic and/or Dual_X tropic HIV thanCCR5-tropic and/or Dual_R tropic and dual-tropic HIV.

In certain embodiments, the step of determining whether the HIVpopulation is a homogeneous or heterogeneous population is performed bydetermining a tropism phenotype for a statistically significant numberof individual viruses infecting the subject. In certain embodiments, atropism phenotype is determined for at least about 100 HIV (molecular orbiological clones representing a single virus). In certain embodiments,a tropism phenotype is determined for at least about 112 HIV. In certainembodiments, a tropism phenotype is determined for at least about 125HIV. In certain embodiments, a tropism phenotype is determined for atleast about 137 HIV. In certain embodiments, a tropism phenotype isdetermined for at least about 150 HIV. In certain embodiments, a tropismphenotype is determined for at least about 162 HIV. In certainembodiments, a tropism phenotype is determined for at least about 175HIV. In certain embodiments, a tropism phenotype is determined for atleast about 187 HIV. In certain embodiments, a tropism phenotype isdetermined for at least about 192 HIV. In certain embodiments, a tropismphenotype is determined for at least about 200 HIV.

In certain embodiments, the step of determining whether the HIVpopulation is a homogeneous or heterogeneous population is performed byevaluating the relative level of infectivity on target cells expressingCD4 and CXCR4 vs CD4 and CCR5. In certain embodiments, the step ofdetermining whether the HIV population is a homogeneous or heterogeneouspopulation is performed by evaluating the inhibition of viralinfectivity by specific co-receptor inhibitors on cells expressing CD4and CXCR4 vs target cells expressing CD4 and CCR5 vs target cellsexpressing CD4 and CXCR4 and CCR5. In certain embodiments, the step ofdetermining whether the HIV population is a homogeneous or heterogeneouspopulation is performed by evaluating the env genotypes of a portion ofthe viruses comprising the population. In some non-limiting embodiments,the method of evaluating the env genotypes is performed according to themethod described in Margulies et al., 2005, Nature 437:376-380. In otherembodiments, the method of evaluating the env genotypes can be performedby sequencing individual viral clones' env genes, or a portion thereof.

In another aspect, the invention provides a method for determiningwhether a subject infected with a dual-mixed tropic population of HIVwould benefit from CXCR4-inhibitor therapy, comprising determiningwhether the HIV population is a homogeneous or heterogeneous populationof HIV, wherein if the subject is infected with a homogeneous populationof HIV and the homogeneous population is Dual_X tropic, the subjectwould benefit from CXCR4-inhibitor therapy; or if the subject isinfected with a heterogeneous population of HIV and the heterogeneouspopulation of HIV comprises substantially more CXCR4-tropic or Dual_Xtropic HIV than total CCR5-tropic, Dual_R tropic, and dual-tropic HIV,the subject would benefit from CXCR4-inhibitor therapy, therebydetermining whether the subject would benefit from CXCR4-inhibitortherapy.

In certain embodiments, the HIV population is homogeneous. In certainembodiments, wherein the HIV population is heterogeneous. In certainembodiments, the step of determining whether the HIV population is ahomogeneous or heterogeneous population is performed by determining atropism phenotype for a statistically significant number of individualviruses infecting the subject.

In still another aspect, the invention provides a method for determiningwhether a subject infected with a dual-mixed tropic population of HIVwould benefit from CCR5-inhibitor therapy, comprising determiningwhether the HIV population is a homogeneous or heterogeneous populationof HIV, wherein if the subject is infected with a homogeneous populationof HIV and the homogeneous population is Dual_R tropic, the subjectwould benefit from CCR5-inhibitor therapy; or if the subject is infectedwith a heterogeneous population of HIV and the heterogeneous populationof HIV comprises substantially more CCR5-tropic or Dual_R tropic HIVthan total CXCR4-tropic, Dual_X tropic, and dual-tropic HIV, the subjectwould benefit from CCR5-inhibitor therapy, thereby determining whetherthe subject would benefit from CCR5-inhibitor therapy.

In certain embodiments, the HIV population is homogeneous. In certainembodiments, the HIV population is heterogeneous. In certainembodiments, the step of determining whether the HIV population is ahomogeneous or heterogeneous population is performed by determining atropism phenotype for a statistically significant number of individualviruses infecting the subject.

In another aspect, the invention provides a method for determiningwhether a subject infected with a dual-mixed tropic population of HIVwould benefit from CCR5-inhibitor therapy and/or CXCR4-inhibitortherapy, comprising determining the primary mechanism of co-receptorusage of the dual-mixed population of HIV, and determining whether thesubject would benefit from CCR5-inhibitor therapy and/or CXCR4-inhibitortherapy.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A: Structure of Envelope Expression and Viral Expression Vectors.

The HIV envelope expression vector (pHIVenv) is modified to acceptenvelope sequences that have been amplified from subject plasma samples.The designations a/b and c/d, refer to restriction endonuclease sitespositioned at the 5′ and 3′ end of the HIV-1 envelope polyprotein(gp160). The HIV expression vector (pHIVlucΔU3) encodes all HIV proteinsexcept the envelope polyprotein. A portion of the envelope gene has beendeleted to accommodate an indicator gene cassette, in this case, fireflyluciferase, that is used to monitor the ability of the virus toreplicate in the presence or absence of anti-viral drugs. The 3′ U3region has been partially deleted to prevent transcription from the 5′LTR in infected cells. Virus produced in this system is limited to asingle round of replication.

FIG. 1B: Cell Based Entry Assay

In this embodiment, drug susceptibility, co-receptor tropism and virusneutralization testing are performed by co-transfecting a host cell withpHIVenv and pHIVlucΔU3. The host cell produces HIV particles that arepseudo-typed with HIV envelope sequences derived from the test virus orsubject sample. Virus particles are collected (˜48 h) after transfectionand are used to infect target cells that express HIV receptors (e.g.CD4) and co-receptors (e.g. CXCR4, CCR5). After infection (˜72 h) thetarget cells are lysed and luciferase activity is measured. HIV mustcomplete one round of replication to successfully infect the target hostcell and produce luciferase activity. If the virus is unable to enterthe target cell, luciferase activity is diminished. This system can beused to evaluate susceptibility to entry inhibitors, receptor andco-receptor tropism, and virus neutralization.

FIG. 2: HIV Envelope Expression Vectors.

HIV envelope sequences are amplified from subject samples and insertedinto expression vectors using restriction endonuclease sites (5′ a/b and3′ c/d). Envelope transcription is driven by the immediate early genepromoter of human cytomegalovirus (CMV). Envelope RNA is polyadenylatedusing an simian virus 40 (SV4O) polyadenylation signal sequence (A+). Anintron located between the CMV promoter and the HIV envelope sequencesis designed to increase envelope mRNA levels in transfected cells.FL-express full-length envelope proteins (gp120, gp41); ΔCT-expressenvelope proteins (gp120, gp21) lacking the C-terminal cytoplasmic taildomain of gp41; +CT-express envelope proteins (gp120, gp41) containing aconstant pre-defined gp41 cytoplasmic tail domain; gp120-express gp120proteins derived from the subject together with a constant pre-definedgp41; and gp41-express a constant pre-defined gp120 together with gp41proteins derived from the subject.

FIG. 3A: Co-Receptor Tropism Screening Assay.

In this figure, the assay is performed using two cell lines. One cellline expresses CD4 and CCR5 (top six panels). The other cell lineexpresses CD4 and CXCR4 (bottom six panels). The assay is performed byinfecting cells with a large number of recombinant virus stocks derivedfrom cells transfected with pHIVenv and pHIVlucΔU3 vectors. The exampleshown represents the analysis of 96 viruses formatted in a 96 well plateinfections are performed in the absence of drug (no drug), or in thepresence of a drug that preferentially inhibits either R5 tropic (CCRinhibitor) or X4 tropic (CXCR4 inhibitor) viruses. Co-receptor tropismis assessed by comparing the amount of luciferase activity produced ineach cell type, both in the presence and absence of drug (see FIG. 3Bfor interpretation of assay results).

FIG. 3B: Determining Co-Receptor Tropism.

In this figure, the results of the assay are interpreted by comparingthe ability of each sample virus to infect (as assessed by the abilityto produce luciferase activity) in cells expressing CD4/CCR5 or cellsexpressing CD4/CXCR4. The ability of a CCR5 or CXCR4 inhibitor tospecifically block infection (as assessed by inhibition of luciferaseactivity) is also evaluated. X4-tropic viruses infect cells expressingCXCR4 but not cells expressing CCR5. Infection of cells expressing CXCR4is blocked by the CXCR4 inhibitor. R5-tropic viruses infect cellsexpressing CCR5 but not cells expressing CXCR4. Infection of cellsexpressing CCR5 is blocked by the CCR5 inhibitor. Infection of cellsexpressing CCR5 by DM pools is blocked by the CCR5 inhibitor andinfection of cells expressing CXCR4 by DM pools is blocked by the CXCR4inhibitor. Individual clones that infect cells expressing CCR5 and CXCR4are classified as dual tropic viruses and can be further classifiedaccording to their ability to infect the cells expressing CXCR4 andCCR5. Dual tropic viruses that infect cells expressing CCR5 efficientlybut enter cells expressing CXCR4 inefficiently are classified as Dual_R.Dual tropic viruses that infect cells expressing CXCR4 efficiently butenter cells expressing CCR5 inefficiently are classified as Dual_X.Non-viable viruses do not replicate in either cells expressing CCR5 orCXCR4.

FIG. 4A: Measuring Entry Inhibitor Susceptibility: Fusion Inhibitor.

In this figure, susceptibility to the fusion inhibitor T-20 isdemonstrated. Cells expressing CD4, CCR5 and CXCR4 were infected in theabsence of T-20 and over a wide range of T-20 concentrations x-axis log10 scale). The percent inhibition of viral replication (y-axis) wasdetermined by comparing the amount of luciferase produced in infectedcells in the presence of T-20 to the amount of luciferase produced inthe absence of T-20. R5 tropic, X4 tropic and dual tropic viruses weretested. Drug susceptibility is quantified by determining theconcentration of T-20 required to inhibit 50% of viral replication(IC₅₀, shown as vertical dashed lines). Viruses with lower IC₅₀ valuesare more susceptible to T-20 than viruses with higher IC₅₀ values.NL4-3: well-characterized X4 tropic strain, JRCSF: well-characterized R5tropic strain, 91US005.11: R5 tropic isolate obtained from the NIH AIDSResearch and Reference Reagent Program (ARRRP), 92HT593.1: Dual tropic(X4R5) isolate obtained from the NIH ARRRP, and 92HT599.24: X4 tropicisolate obtained from the NIH ARRRP.

FIG. 4B: Measuring Entry Inhibitor Susceptibility: Drug ResistanceMutations.

In this figure, reduced susceptibility to the fusion inhibitor T-20conferred by specific drug resistance mutations in the gp41 envelopeprotein is demonstrated. Cells expressing CD4, CCR5 and CXCR4 wereinfected in the absence of T-20 and over a wide range of T-20concentrations x-axis log 10 scale). The percent inhibition of viralreplication (y-axis) was determined by comparing the amount ofluciferase produced in infected cells in the presence of T-20 to theamount of luciferase produced in the absence of T-20. Isogenic virusescontaining one or two specific mutations in the gp41 transmembraneenvelope, protein were tested (highlighted in red in the figure legend).Drug susceptibility is quantified by determining the concentration ofT-20 required to inhibit 50% of viral replication (IC₅₀, shown asvertical dashed lines). Viruses with lower IC₅₀ values are moresusceptible to T-20 than viruses with higher IC₅₀ values. No mutation(wildtype sequence): GIV; Single mutations: GIV, DIM, SIV; Doublemutations: DIM, SIM, DTV.

FIG. 5: Different Dual Subtypes Exhibit Different Patterns of Inhibitionto Co-Receptor Inhibitors.

FIG. 5 presents a diagram showing different inhibition patters of dualtropic viruses that are Dual_R-tropic, Dual_X-tropic, or Dual tropicwhen entering cells expressing CD4 and CXCR4, CCR5, or both CXCR4 andCCR5.

FIG. 6: Dual Tropic Clones are Incompletely Inhibited on CellsExpressing CD4 and CXCR4 and CCR5.

FIG. 6 presents a diagram showing relative inhibition by CCR5 inhibitorsand CXCR4 inhibitors of entry to cells expressing CD4 and CXCR4, CD4 andCCR5, or CD4 and both CXCR4 and CCR5.

FIG. 7: Infectious Clones Exhibit Similar Patterns of Inhibition byCo-Receptor Inhibitors on PBMCs.

FIG. 7 presents a diagram showing relative block by CCR5-inhibitors andCXCR4-inhibitors to infection of peripheral blood monocytic cells(PBMCs).

FIG. 8: Suppression of CXCR4-Tropic Viruses following CXCR4 InhibitorTherapy.

FIG. 8 presents a table showing patients experiencing suppression andnon-suppression of CXCR4-tropic viruses following CXCR4-inhibitortherapy.

FIG. 9: Suppression of CXCR4-Tropic Virus Depends on the ViralComposition at Baseline.

FIG. 9 presents a table showing that suppression of CXCR4-tropic virusdepends on the relative proportion of CCR5-tropic, CXCR4-tropic, anddual-tropic virus in the viral population at baseline prior totreatment.

5. DEFINITIONS

As used herein, the following terms shall have the following meanings:

A “phenotypic assay” is a functional test that measures a phenotype of aparticular virus, such as, for example, HIV, or a population of viruses,such as, for example, the population of HIV infecting a subject. Thephenotypes of a virus or virus population that can be measured include,but are not limited to, drug or antibody resistance or susceptibility,replication capacity, infectivity, membrane fusion, virion assembly,virion maturation, virion egress, pathogenesis, or cytopathogenicity.

A “genotypic assay” is an assay that determines a genotype of anorganism, a part of an organism, a population of organisms, a gene, apart of a gene, or a population of genes. Typically, a genotypic assayinvolves determination of the nucleic acid sequence of the relevant geneor genes. Such assays are frequently performed in HIV to establish, forexample, whether certain mutations are associated with drug resistanceor resistance or altered replication capacity are present. Theinterpretation of genotypic assay results, based either on rules oralgorithms, are often used to predict the phenotype of a virus or viruspopulation, but are themselves not functional assays.

The term “% sequence identity” is used interchangeably herein with theterm “% identity” and refers to the level of amino acid sequenceidentity between two or more peptide sequences or the level ofnucleotide sequence identity between two or more nucleotide sequences,when aligned using a sequence alignment program. For example, as usedherein, 80% identity means the same thing as 80% sequence identitydetermined by a defined algorithm, and means that a given sequence is atleast 80% identical to another length of another sequence. Exemplarylevels of sequence identity include, but are not limited to, 60, 70, 80,85, 90, 95, 98% or more sequence identity to a given sequence.

The term “% sequence homology” is used interchangeably herein with theterm “% homology” and refers to the level of amino acid sequencehomology between two or more peptide sequences or the level ofnucleotide sequence homology between two or more nucleotide sequences,when aligned using a sequence alignment program. For example, as usedherein, 80% homology means the same thing as 80% sequence homologydetermined by a defined algorithm, and accordingly a homologue of agiven sequence has greater than 80% sequence homology over a length ofthe given sequence. Exemplary levels of sequence homology include, butare not limited to, 60, 70, 80, 85, 90, 95, 98% or more sequencehomology to a given sequence.

Exemplary computer programs which can be used to determine identitybetween two sequences include, but are not limited to, the suite ofBLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN,publicly available on the Internet at the NCBI website. See alsoAltschul et al., 1990, J. Mol. Biol. 215:403-10 (with special referenceto the published default setting, i.e., parameters w=4, t=17) andAltschul et al., 1997, Nucleic Acids Res., 25:3389-3402. Sequencesearches are typically carried out using the BLASTP program whenevaluating a given amino acid sequence relative to amino acid sequencesin the GenBank Protein Sequences and other public databases. The BLASTXprogram is preferred for searching nucleic acid sequences that have beentranslated in all reading frames against amino acid sequences in theGenBank Protein Sequences and other public databases. Both BLASTP andBLASTX are run using default parameters of an open gap penalty of 11.0,and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix.See id.

A preferred alignment of selected sequences in order to determine “%identity” between two or more sequences, is performed using for example,the CLUSTAL-X program, operated with default parameters, including anopen gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM30 similarity matrix.

“Polar Amino Acid” refers to a hydrophilic amino acid having a sidechain that is uncharged at physiological pH, but which has at least onebond in which the pair of electrons shared in common by two atoms isheld more closely by one of the atoms. Genetically encoded polar aminoacids include Asn (N), Gln (O) Ser (S) and Thr (T).

“Nonpolar Amino Acid” refers to a hydrophobic amino acid having a sidechain that is uncharged at physiological pH and which has bonds in whichthe pair of electrons shared in common by two atoms is generally heldequally by each of the two atoms (i.e., the side chain is not polar).Genetically encoded nonpolar amino acids include Ala (A), Gly (G), Ile(I), Leu (L), Met (M) and Val (V).

“Hydrophilic Amino Acid” refers to an amino acid exhibiting ahydrophobicity of less than zero according to the normalized consensushydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol.179:125-142. Genetically encoded hydrophilic amino acids include Arg(R), Asn (N), Asp (D), Glu (E), Gln (O), H is (H), Lys (K), Ser (S) andThr (T).

“Hydrophobic Amino Acid” refers to an amino acid exhibiting ahydrophobicity of greater than zero according to the normalizedconsensus hydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol.179:125-142. Genetically encoded hydrophobic amino acids include Ala(A), Gly (G), Ile (I), Leu (L), Met (M), Phe (F), Pro (P), Trp (W), Tyr(Y) and Val (V).

“Acidic Amino Acid” refers to a hydrophilic amino acid having a sidechain pK value of less than 7. Acidic amino acids typically havenegatively charged side chains at physiological pH due to loss of ahydrogen ion. Genetically encoded acidic amino acids include Asp (D) andGlu (E).

“Basic Amino Acid” refers to a hydrophilic amino acid having a sidechain pK value of greater than 7. Basic amino acids typically havepositively charged side chains at physiological pH due to associationwith a hydrogen ion. Genetically encoded basic amino acids include Arg(R), His (H) and Lys (K).

A “mutation” is a change in an amino acid sequence or in a correspondingnucleic acid sequence relative to a reference nucleic acid orpolypeptide. For embodiments of the invention comprising HIV protease orreverse transcriptase, the reference nucleic acid encoding protease,reverse transcriptase, or envelope is the protease, reversetranscriptase, or envelope coding sequence, respectively, present inNL4-3 HIV (GenBank Accession No. AF324493). Likewise, the referenceprotease, reverse transcriptase, or envelope polypeptide is that encodedby the NL4-3 HIV sequence. Although the amino acid sequence of a peptidecan be determined directly by, for example, Edman degradation or massspectroscopy, more typically, the amino sequence of a peptide isinferred from the nucleotide sequence of a nucleic acid that encodes thepeptide. Any method for determining the sequence of a nucleic acid knownin the art can be used, for example, Maxam-Gilbert sequencing (Maxam etal., 1980, Methods in Enzymology 65:499), dideoxy sequencing (Sanger etal., 1977, Proc. Natl. Acad. Sci. USA 74:5463) or hybridization-basedapproaches (see e.g., Sambrook et al., 2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, 3^(rd) ed., NY; andAusubel et al., 1989, Current Protocols in Molecular Biology, GreenePublishing Associates and Wiley Interscience, NY).

A “mutant” is a virus, gene or protein having a sequence that has one ormore changes relative to a reference virus, gene or protein.

The terms “peptide,” “polypeptide” and “protein” are usedinterchangeably throughout.

The term “wild-type” refers to a viral genotype that does not comprise amutation known to be associated with drug resistance.

The terms “polynucleotide,” “oligonucleotide” and “nucleic acid” areused interchangeably throughout.

A “dual-mixed tropic” population of HIV, as used herein, refers to apopulation of HIV viruses that collectively have the ability to entercells expressing CD4 and CCR5 or cells expressing CD4 and CXCR4.Exemplary dual-mixed tropic populations of HIV include, for example, ahomogeneous population of dual tropic HIV, a heterogeneous population ofHIV that comprises viruses that can enter cells expressing CD4 and CCR5and viruses that can enter cells expressing CD4 and CXCR4, aheterogeneous population of HIV that comprises viruses that can entercells expressing CD4 and CCR5 and viruses that can enter cellsexpressing CD4 and either CCR5 or CXCR4, and the like.

A “homogeneous” population of viruses is a population of viruses whosemembers have the same or similar phenotype when a statisticallysignificant number of members' phenotypes are assessed. One example of aphenotype according to this definition is a tropism phenotype.

A “heterogeneous” population of viruses is a population of viruses thatcomprises members having at least two different phenotypes when astatistically significant number of members' phenotypes are assessed.One example of a phenotype according to this definition is a tropismphenotype.

An “R5 tropic” or “CCR5-tropic” virus is a single virus or viruspopulation that can normally enter cells expressing CD4 and CCR5, but donot normally enter cells expressing CD4 and CXCR4.

An “X4 tropic” or “CXCR4-tropic” virus is a single virus or viruspopulation that can normally enter cells expressing CD4 and CXCR4, butdo not normally enter cells expressing CD4 and CCR5.

A “dual tropic” or “dual-tropic” virus or viral population is a singlevirus or virus population that can enter cells expressing CD4 and eitherCCR5 or CXCR4 that does not have a significantly greater (e.g., 10-fold100-fold, 1000-fold, or greater) ability to enter cells co-expressingCXCR4 relative to its ability to enter cells co-expressing CCR5, or viceversa.

A Dual_R-tropic virus (also referred to herein as a primarilyCCR5-tropic virus) is a single virus that can enter cells expressing CD4and either CCR5 or CXCR4, but has a significantly greater (e.g.,10-fold, 100-fold, 1000-fold, or greater) ability to enter cellsco-expressing CCR5 relative to its ability to enter cells co-expressingCXCR4.

A Dual_X-tropic virus (also referred to herein as a primarilyCXCR4-tropic virus) is a single virus that can enter cells expressingCD4 and either CCR5 or CXCR4, but has a significantly greater (e.g.,10-fold, 100-fold, 1000-fold, or greater) ability to enter cellsco-expressing CXCR4 relative to its ability to enter cells co-expressingCCR5.

“Substantially more,” in the context of the relative phenotypes in aviral population, refers to a proportion of at least about 90% of onephenotype to about 10% or less of one or more other phenotypes. Forexample, “subtantially more” CXCR4-tropic viruses than CCR5 tropicviruses means that, of a total population containing both kinds ofviruses, at least about 90% or greater of the viruses are CXCR4 tropicand about 10% or less of the viruses are CCR5 tropic.

“Relatively comparable,” in the context of the relative phenotypes in aviral population, refers to a proportion of less than about 90% of onephenotype to 10% or greater of one or more additional phenotypes. Forexample, “relatively comparable” amounts of CXCR4-tropic and CCR5-tropicviruses means that less than 90% of the total population of HIV isCXCR4-tropic and less than 90% of the total population of HIV isCCR5-tropic.

A “benefit” from a co-receptor inhibitor therapy refers to any desirableclinical or therapeutic endpoint or indicator normally associated withHIV antiviral therapy, and can include, but is not limited to, areduction in viral load, an inhibition of replication in a particularcompartment or target cell type in the body that results in preservationor restoration of that compartment or cell type, a preservation orrestoration of compartment or target cell type that results in improvedimmunologic function (for example, CD4+T-cells) or specific or generalhealth of the patient (for example, suppression of replication in theCNS).

The term “about,” as used herein, unless otherwise indicated, refers toa value that is no more than 10% above or below the value being modifiedby the term. For example, the term “about 5 μg/kg” means a range of from4.5 μg/kg to 5.5 μg/kg. As another example, “about 1 hour” means a rangeof from 48 minutes to 72 minutes.

6. DETAILED DESCRIPTION OF THE INVENTION

In certain aspects, the invention provides methods for determiningwhether a subject infected with a dual-mixed tropic population of humanimmunodeficiency viruses (HIV) would benefit from CXCR4-inhibitor orCCR5-inhibitor therapy. The methods are useful, for example, to guidetherapeutic decisions in treatment subjects infected with HIV, includedbut not limited to the initiation of treatment or a change in treatment.Change in treatment may be desired, for example, to restore suppressionof virus replication (e.g. after treatment failure) or to minimize oreliminate undesirable or toxic side affects (during successfultreatment). Other uses of such methods will be apparent to those ofskill in the art.

6.1 Determining Viral Tropism Phenotypes

In one aspect, the invention provides a method for determining whether asubject infected with a dual-mixed tropic population of HIV wouldbenefit from CXCR4-inhibitor or CCR5-inhibitor therapy. In such methods,the tropism of the HIV population or the individual HIV of thepopulation can be determined using any method known to one skilled inthe art without limitation. In one embodiment, the tropism is determinedusing a single cell infectivity assay as described in U.S. Pat. Nos.7,169,551 and 7,097,970 and U.S. Patent Application Publication Nos.20060183110 and 20060160185. In another embodiment, tropism isdetermined by performing an infectivity assay using PBMCs in thepresence of one or more co-receptor inhibitors.

6.2 Determining Viral Genotypes

Viral genotypes can be detected by utilizing any suitable techniqueknown to one of skill in the art without limitation. Viral DNA or RNAcan be used as the starting point for such assay techniques, and may beisolated according to standard procedures which are well known to thoseof skill in the art.

The determination of specific nucleic acid sequences, such as in aparticular region of the env gene, can be accomplished by a variety ofmethods including, but not limited to,restriction-fragment-length-polymorphism detection based onallele-specific restriction-endonuclease cleavage (Kan and Dozy, 1978,Lancet ii:910-912), mismatch-repair detection (Faham and Cox, 1995,Genome Res 5:474-482), binding of MutS protein (Wagner et al., 1995,Nucl Acids Res 23:3944-3948), denaturing-gradient gel electrophoresis(Fisher et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:1579-83),single-strand-conformation-polymorphism detection (Orita et al., 1983,Genomics 5:874-879), RNAase cleavage at mismatched base-pairs (Myers etal., 1985, Science 230:1242), chemical (Cotton et al., 1988, Proc. Natl.Acad. Sci. U.S.A. 85:4397-4401) or enzymatic (Youil et al., 1995, Proc.Natl. Acad. Sci. U.S.A. 92:87-91) cleavage of heteroduplex DNA, methodsbased on oligonucleotide-specific primer extension (Syvanen et al.,1990, Genomics 8:684-692), genetic bit analysis (Nikiforov et al., 1994,Nucl Acids Res 22:4167-4175), oligonucleotide-ligation assay (Landegrenet al., 1988, Science 241:1077), oligonucleotide-specific ligation chainreaction (“LCR”) (Barrany, 1991, Proc. Natl. Acad. Sci. U.S.A.88:189-193), gap-LCR (Abravaya et al., 1995, Nucl Acids Res 23:675-682),radioactive or fluorescent DNA sequencing using standard procedures wellknown in the art, and peptide nucleic acid (PNA) assays (Orum et al.,1993, Nucl. Acids Res. 21:5332-5356; Thiede et al., 1996, Nucl. AcidsRes. 24:983-984).

In addition, viral DNA or RNA may be used in hybridization oramplification assays to detect abnormalities involving gene structure,including point mutations, insertions, deletions and genomicrearrangements. Such assays may include, but are not limited to,Southern analyses (Southern, 1975, J. Mol. Biol. 98:503-517), singlestranded conformational polymorphism analyses (SSCP) (Orita et al.,1989, Proc. Natl. Acad. Sci. USA 86:2766-2770), and PCR analyses (U.S.Pat. Nos. 4,683,202; 4,683,195; 4,800,159; and 4,965,188; PCRStrategies, 1995 Innis et al. (eds.), Academic Press, Inc.).

Such diagnostic methods can involve for example, contacting andincubating the viral nucleic acids with one or more labeled nucleic acidreagents including recombinant DNA molecules, cloned genes or degeneratevariants thereof, under conditions favorable for the specific annealingof these reagents to their complementary sequences. Preferably, thelengths of these nucleic acid reagents are at least 15 to 30nucleotides. After incubation, all non-annealed nucleic acids areremoved from the nucleic acid molecule hybrid. The presence of nucleicacids which have hybridized, if any such molecules exist, is thendetected. Using such a detection scheme, the nucleic acid from the viruscan be immobilized, for example, to a solid support such as a membrane,or a plastic surface such as that on a microtiter plate or polystyrenebeads. In this case, after incubation, non-annealed, labeled nucleicacid reagents of the type described above are easily removed. Detectionof the remaining, annealed, labeled nucleic acid reagents isaccomplished using standard techniques well-known to those in the art.The gene sequences to which the nucleic acid reagents have annealed canbe compared to the annealing pattern expected from a normal genesequence in order to determine viral env gene sequences.

These techniques can easily be adapted to provide high-throughputmethods for determining viral sequences. For example, a gene array fromAffymetrix (Affymetrix, Inc., Sunnyvale, Calif.) can be used to rapidlyidentify genotypes of a large number of individual viruses. Affymetrixgene arrays, and methods of making and using such arrays, are describedin, for example, U.S. Pat. Nos. 6,551,784, 6,548,257, 6,505,125,6,489,114, 6,451,536, 6,410,229, 6,391,550, 6,379,895, 6,355,432,6,342,355, 6,333,155, 6,308,170, 6,291,183, 6,287,850, 6,261,776,6,225,625, 6,197,506, 6,168,948, 6,156,501, 6,141,096, 6,040,138,6,022,963, 5,919,523, 5,837,832, 5,744,305, 5,834,758, and 5,631,734,each of which is hereby incorporated by reference in its entirety.

Alternately, the genotypes of many viruses can be determinedsimultaneously using or adapting the methods described in Margulies etal., 2005, Nature 437:376-380 and in U.S. Pat. Nos. 6,956,114 and6,902,921.

In addition, Ausubel et al., eds., Current Protocols in MolecularBiology, 2002, Vol. 4, Unit 25B, Ch. 22, which is hereby incorporated byreference in its entirety, provides further guidance on construction anduse of a gene array for determining the genotypes of a large number ofviral isolates. Finally, U.S. Pat. Nos. 6,670,124; 6,617,112; 6,309,823;6,284,465; and 5,723,320, each of which is incorporated by reference inits entirety, describe related array technologies that can readily beadapted for rapid identification of a large number of viral genotypes byone of skill in the art.

Alternative diagnostic methods for the detection of gene specificnucleic acid molecules may involve their amplification, e.g., by PCR(U.S. Pat. Nos. 4,683,202; 4,683,195; 4,800,159; and 4,965,188; PCRStrategies, 1995 Innis et al. (eds.), Academic Press, Inc.), followed bythe detection of the amplified molecules using techniques well known tothose of skill in the art. The resulting amplified sequences can becompared to those which would be expected if the nucleic acid beingamplified contained only normal copies of the respective gene in orderto viral env gene sequences.

Additionally, the nucleic acid can be sequenced by any sequencing methodknown in the art. For example, the viral DNA can be sequenced by thedideoxy method of Sanger et al., 1977, Proc. Natl. Acad. Sci. USA74:5463, as further described by Messing et al., 1981, Nuc. Acids Res.9:309, or by the method of Maxam et al., 1980, Methods in Enzymology65:499. See also the techniques described in Sambrook et al., 2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,3^(rd) ed., NY; and Ausubel et al., 1989, Current Protocols in MolecularBiology, Greene Publishing Associates and Wiley Interscience, NY.

Antibodies directed against the viral gene products, i.e., viralproteins or viral peptide fragments can also be used to detectparticular envelope protein sequences in the viral proteins.Alternatively, the viral protein or peptide fragments of interest can besequenced by any sequencing method known in the art in order to yieldthe amino acid sequence of the protein of interest. An example of such amethod is the Edman degradation method which can be used to sequencesmall proteins or polypeptides. Larger proteins can be initially cleavedby chemical or enzymatic reagents known in the art, for example,cyanogen bromide, hydroxylamine, trypsin or chymotrypsin, and thensequenced by the Edman degradation method.

6.3 Computer-Implemented Methods

In another aspect, the present invention provides computer-implementedmethods for performing a method of the invention. In such embodiments,the methods of the invention are adapted to take advantage of theprocessing power of modern computers. One of skill in the art canreadily adapt the methods in such a manner. Such methods generallycomprise inputting data regarding a viral population phenotype and/or avirus phenotype into a computer-readable memory, inputting a correlationbetween the population or virus's phenotype and benefit to a subject fortherapy with a co-receptor inhibitor, and determining whether thesubject will benefit from such therapy.

In yet another aspect, the invention provides a computer-readable mediumthat comprises data generated according a method of the invention. Incertain embodiments, the computer-readable medium is a random-accessmemory. In certain embodiments, the computer-readable medium is a fixeddisk. In certain embodiments, the computer-readable medium is a floppydisk. In certain embodiments, the computer-readable medium is a portablememory device, such as, e.g., a USB key or an iPod.™.

In still another aspect, the invention provides an article ofmanufacture that comprises computer-readable instructions for performinga method of the invention. In certain embodiments, the article ofmanufacture is a random-access memory. In certain embodiments, thearticle of manufacture is a fixed disk. In certain embodiments, thearticle of manufacture is a floppy disk. In certain embodiments, thearticle of manufacture is a portable memory device, such as, e.g., a USBkey or an iPod.™.

In yet another aspect, the invention provides a computer-readable mediumthat comprises data generated according to method of the invention andcomputer-readable instructions for performing a method of the invention.In certain embodiments, the computer-readable medium is a random-accessmemory. In certain embodiments, the computer-readable medium is a fixeddisk. In certain embodiments, the computer-readable medium is a floppydisk. In certain embodiments, the computer-readable medium is a portablememory device, such as, e.g., a USB key or an iPod.™.

In yet another aspect, the invention provides a computer system that isconfigured to perform a method of the invention.

6.4 Viruses and Viral Samples

The co-receptor usage phenotypes and susceptibility to co-receptorinhibitor phenotypes of viruses and viral populations can be determinedfrom a viral sample obtained by any means known in the art for obtainingviral samples. Such methods include, but are not limited to, obtaining aviral sample from a human or an animal infected with the virus orobtaining a viral sample from a viral culture. In one embodiment, theviral sample is obtained from a human individual infected with thevirus. The viral sample could be obtained from any part of the infectedindividual's body or any secretion expected to contain the virus.Examples of such parts include, but are not limited to blood, serum,plasma, sputum, lymphatic fluid, semen, vaginal mucus and samples ofother bodily fluids. In a preferred embodiment, the sample is a blood,serum or plasma sample.

In another embodiment, the phenotypes are determined from a virus orviral population that can be obtained from a culture. In someembodiments, the culture can be obtained from a laboratory. In otherembodiments, the culture can be obtained from a collection, for example,the American Type Culture Collection.

In certain embodiments, the viral or viral population phenotype isdetermined from a derivative of a virus. In one embodiment, thederivative of the virus is not itself pathogenic. In another embodiment,the derivative of the virus is a plasmid-based system, whereinreplication of the plasmid or of a cell transfected with the plasmid isaffected by the presence or absence of the selective pressure, such thatmutations are selected that increase resistance to the selectivepressure. In some embodiments, the derivative of the virus comprises thenucleic acids or proteins of interest, for example, those nucleic acidsor proteins to be targeted by an anti-viral treatment. In oneembodiment, the genes of interest can be incorporated into a vector.See, e.g., U.S. Pat. Nos. 7,169,551 and 7,097,970 and U.S. PatentApplication Publication Nos. 20060183110 and 20060160185, each of whichis incorporated herein by reference. In certain embodiments, the genescan be those that encode envelope protein (gp160).

In another embodiment, the intact virus need not be used. Instead, apart of the virus incorporated into a vector can be used. Preferablythat part of the virus is used that is targeted by an anti-viral drug.

In another embodiment, the viral or viral population phenotype isdetermined in a genetically modified virus. The virus can be geneticallymodified using any method known in the art for genetically modifying avirus. For example, the virus can be grown for a desired number ofgenerations in a laboratory culture. In one embodiment, no selectivepressure is applied (i.e., the virus is not subjected to a treatmentthat favors the replication of viruses with certain characteristics),and new mutations accumulate through random genetic drift. In anotherembodiment, a selective pressure is applied to the virus as it is grownin culture (i.e., the virus is grown under conditions that favor thereplication of viruses having one or more characteristics). In oneembodiment, the selective pressure is an anti-viral treatment. Any knownanti-viral treatment can be used as the selective pressure.

In certain embodiments, the virus is HIV and the selective pressure is aNNRTI. In another embodiment, the virus is HIV-1 and the selectivepressure is a NNRTI. Any NNRTI can be used to apply the selectivepressure. Examples of NNRTIs include, but are not limited to,nevirapine, delavirdine and efavirenz. By treating HIV cultured in vitrowith a NNRTI, one can select for mutant strains of HIV that have anincreased resistance to the NNRTI. The stringency of the selectivepressure can be manipulated to increase or decrease the survival ofviruses not having the selected-for characteristic.

In other embodiments, the virus is HIV and the selective pressure is aNRTI. In another embodiment, the virus is HIV-1 and the selectivepressure is a NRTI. Any NRTI can be used to apply the selectivepressure. Examples of NRTIs include, but are not limited to, AZT, ddI,ddC, d4T, 3TC, abacavir, and tenofovir. By treating HIV cultured invitro with a NRTI, one can select for mutant strains of HIV that have anincreased resistance to the NRTI. The stringency of the selectivepressure can be manipulated to increase or decrease the survival ofviruses not having the selected-for characteristic.

In still other embodiments, the virus is HIV and the selective pressureis a PI. In another embodiment, the virus is HIV-1 and the selectivepressure is a PI. Any PI can be used to apply the selective pressure.Examples of PIs include, but are not limited to, saquinavir, ritonavir,indinavir, nelfinavir, amprenavir, lopinavir and atazanavir. By treatingHIV cultured in vitro with a PI, one can select for mutant strains ofHIV that have an increased resistance to the PI. The stringency of theselective pressure can be manipulated to increase or decrease thesurvival of viruses not having the selected-for characteristic.

In still other embodiments, the virus is HIV and the selective pressureis an entry inhibitor. In another embodiment, the virus is HIV-1 and theselective pressure is an entry inhibitor. Any entry inhibitor can beused to apply the selective pressure. An example of a entry inhibitorincludes, but is not limited to, fusion inhibitors such as, for example,enfuvirtide. Other entry inhibitors include co-receptor inhibitors, suchas, for example, AMD3100 (Anormed). Such co-receptor inhibitors caninclude any compound that interferes with an interaction between HIV anda co-receptor, e.g., CCR5 or CRCX4, without limitation. Still otherentry inhibitors include UK-427857 (Pfizer), TNX-355 (Tanox Inc.),AMD-070 (AnorMED), Pro 140 (Progenics), FP-21399 (EMD Lexigen),BMS-488043 (Bristol-Myers Squibb), and GSK-873,140 (GlaxoSmithKline). Bytreating HIV cultured in vitro with an entry inhibitor, one can selectfor mutant strains of HIV that have an increased resistance to the entryinhibitor. The stringency of the selective pressure can be manipulatedto increase or decrease the survival of viruses not having theselected-for characteristic.

In another aspect, a mutation associated an altered tropism phenotypecan be made by mutagenizing a virus, a viral genome, or a part of aviral genome. Any method of mutagenesis known in the art can be used forthis purpose. In certain embodiments, the mutagenesis is essentiallyrandom. In certain embodiments, the essentially random mutagenesis isperformed by exposing the virus, viral genome or part of the viralgenome to a mutagenic treatment. In another embodiment, a gene thatencodes a viral protein that is the target of an anti-viral therapy ismutagenized. Examples of essentially random mutagenic treatmentsinclude, for example, exposure to mutagenic substances (e.g., ethidiumbromide, ethylmethanesulphonate, ethyl nitroso urea (ENU) etc.)radiation (e.g., ultraviolet light), the insertion and/or removal oftransposable elements (e.g., Tn5, Tn10), or replication in a cell, cellextract, or in vitro replication system that has an increased rate ofmutagenesis. See, e.g., Russell et al., 1979, Proc. Nat. Acad. Sci. USA76:5918-5922; Russell, W., 1982, Environmental Mutagens and Carcinogens:Proceedings of the Third International Conference on EnvironmentalMutagens. One of skill in the art will appreciate that while each ofthese methods of mutagenesis is essentially random, at a molecularlevel, each has its own preferred targets.

In another aspect, a viral population or virus phenotype can bedetermined in an HIV or HIV derivative made using site-directedmutagenesis. Any method of site-directed mutagenesis known in the artcan be used (see e.g., Sambrook et al., 2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, 3^(rd) ed., NY; andAusubel et al., 1989, Current Protocols in Molecular Biology, GreenePublishing Associates and Wiley Interscience, NY). See, e.g., Sarkar andSommer, 1990, Biotechniques, 8:404-407. The site directed mutagenesiscan be directed to, e.g., a particular gene or genomic region, aparticular part of a gene or genomic region, or one or a few particularnucleotides within a gene or genomic region. In one embodiment, the sitedirected mutagenesis is directed to a viral genomic region, gene, genefragment, or nucleotide based on one or more criteria. In oneembodiment, a gene or a portion of a gene is subjected to site-directedmutagenesis because it encodes a protein that is known or suspected tobe a target of an anti-viral therapy, e.g., the gene encoding the HIVreverse transcriptase. In another embodiment, a portion of a gene, orone or a few nucleotides within a gene, are selected for site-directedmutagenesis. In one embodiment, the nucleotides to be mutagenized encodeamino acid residues that are known or suspected to interact with ananti-viral compound. In another embodiment, the nucleotides to bemutagenized encode amino acid residues that are known or suspected to bemutated in viral strains that are resistant or susceptible orhypersusceptible to one or more antiviral agents. In another embodiment,the mutagenized nucleotides encode amino acid residues that are adjacentto or near in the primary sequence of the protein residues known orsuspected to interact with an anti-viral compound or known or suspectedto be mutated in viral strains that are resistant or susceptible orhypersusceptible to one or more antiviral agents. In another embodiment,the mutagenized nucleotides encode amino acid residues that are adjacentto or near to in the secondary, tertiary or quaternary structure of theprotein residues known or suspected to interact with an anti-viralcompound or known or suspected to be mutated in viral strains having analtered replication capacity. In another embodiment, the mutagenizednucleotides encode amino acid residues in or near the active site of aprotein that is known or suspected to bind to an anti-viral compound.

7. EXAMPLES

The following examples are presented to further illustrate and explainthe present invention and should not be taken as limiting in any regard.Certain of these experiments were also described in U.S. Pat. Nos.7,169,551 and 7,097,970 and U.S. Patent Application Publication Nos.20060183110 and 20060160185, each of which is incorporated by referencein its entirety.

7.1 Example 1

Measuring Phenotypic Drug Susceptibility to Inhibitors of HIV-1 Entry

This example provides a means and method for accurately and reproduciblymeasuring susceptibility to inhibitors of HIV-1 attachment and entry(heretofore collectively referred to as entry). Based on this example,the means and method for measuring susceptibility to inhibitors of HIV-1entry can be adapted to other viruses, including, but not limited toother lentiviruses (e.g. HIV-2), other retroviruses (e.g. HTLV-1 and 2),hepadnaviruses (human hepatitis B virus), flaviviruses (human hepatitisC virus) and herpesviruses human cytomegalovirus). This example furtherprovides a means and method for measuring alterations (increases anddecreases) in susceptibility to entry inhibitors.

Measurements of entry inhibitor susceptibility are carried out usingadaptations of the means and methods for phenotypic drug susceptibilityand resistance tests described in U.S. Pat. No. 5,837,464 (InternationalPublication Number WO 97/27319) which is hereby incorporated byreference.

One vector, an example of the envelope expression vector, (pHIVenv) isdesigned to express the envelope polyprotein (gp160) encoded by subjectderived HIV envelope sequences (FIG. 1). Gp 160 is subsequently cleavedby a cellular protease to generate the surface (gp120SU) andtransmembrane (gp41™) subunits that comprise the envelope protein on thesurface of HIV-1 virus particles. A second vector, an example of theviral expression vector, (either pHIVluc or pHIVlucΔU3) is designed toexpress genomic and subgenomic viral RNAs and all HIV proteins exceptthe envelope polyprotein (FIGS. 1A-1B).

In this application, patient-derived segment(s) correspond to the codingregion (−2.5 KB) of the HIV-1 envelope polyprotein (gp160) and representeither (a) envelope sequences amplified by the reversetranscription-polymerase chain reaction method CRT-PCR) using viral RNAisolated from virus derived from HIV-infected individuals, or (b)envelope sequences derived from molecular clones of HIV-1 that containspecific mutations introduced by site directed mutagenesis of a parentalmolecular clone (typically NL4-3).

Isolation of viral RNA was performed using standard procedures (e.g.RNAgents Total RNA Isolation System, Promega, Madison Wis. or RNAzo1,Tel-Test, Friendswood, Tex.). The RT-PCR protocol was divided into twosteps. A retroviral reverse transcriptase [e.g. Superscript II(Invitrogen, Life Technologies) Moloney MuLV reverse transcriptase(Roche Molecular Systems, Inc., Branchburg, N.J.), or avianmyeloblastosis virus (AMV) reverse transcriptase, (Boehringer Mannheim,Indianapolis, Ind.)] was used to copy viral RNA into first strand cDNA.The cDNA was then amplified to high copy number using a thermostable DNApolymerase [e.g. Taq (Roche Molecular Systems, Inc., Branchburg, N.J.),Tth (Roche Molecular Systems, Inc., Branchburg, N.J.), PrimeZyme(isolated from Thermus brockianus, Biometra, Gottingen, Germany)] or acombination of thermostable polymerases as described for the performanceof “long PCR” (Barnes, W. M., (1994) Proc. Natl. Acad. Sci, USA 91,2216-2220) [e.g. Expand High Fidelity PCR System (Taq+Pwo), (BoehringerMannheim. Indianapolis, Ind.) OR GeneAmp XL PCR kit (Tth+Vent), (RocheMolecular Systems, Inc., Branchburg, N.J.), Advantage-2, (CloneTech).

Oligo-dT was used for reverse transcription of viral RNA into firststrand cDNA. Envelope PCR primers, forward primer Xho/Pin and reverseprimer Mlu/Xba (Table 3) were used to amplify the patient-derivedsegments. These primers are designed to amplify the −2.5 kB envelopegene encoding the gp160 envelope polyprotein, while introducing Xho Iand Pin AI recognition sites at the 5′ end of the PCR amplificationproduct, and Mlu I and Xba I sites at the 3′ end of the PCRamplification product.

Subject derived segments (2.5 kB envelope sequence amplificationproduct) were inserted into HIV-1 envelope expression vectors usingrestriction endonuclease digestion, DNA ligation and bacterialtransformation methods as described in U.S. Pat. No. 5,837,464(International Publication Number WO 97/27319), with minor adaptations.The ˜2.5 kB amplification product was digested with either Xho I or PinAI at the 5′ end and either Mlu I or Xba I at the 3′ end. The resultingdigestion products were ligated, using DNA ligase, into the 5′ Xho I/PinAI and 3′ Mlu I/Xba I sites of modified pCXAS or pCXAT expressionvectors. The construction of the pCXAS and pCXAT vectors has beendescribed in U.S. Pat. No. 5,837,464 (International Publication NumberWO 97/27319)). Modified pCXAS and pCXAT vectors contain a Pin AIrestriction site in addition to the Xho I, MluI and Xba I restrictionsites that exist in pCXAS and pCXAT. The Pin AI site was introducedbetween the Xho I and Mlu I sites by site directed mutagenesis, suchthat the four sites are located 5′ to 3′ in the following order; Xho I,Pin AI, Mlu I and Xba I. In a preferred embodiment, the 2.5 kBamplification products were digested with Pin AI and Mlu I and ligatedinto the 5′ Pin AI site and the 3′ Mlu I site of the modified pCXASexpression vector. Ligation reaction products were used to transform E.coli. Following a 24-36 h incubation period at 30-37.degree. C., theexpression vector plasmid DNA was purified from the E. coli cultures. Toensure that expression vector preparations adequately represents the HIVquasi-species present in the serum of a given subject, many (>100)independent E. coli transformants were pooled and used for thepreparations of pHIVenv plasmid DNA. Vectors that are assembled in thismanner for the purposes of expressing subject virus derived envelopeproteins are collectively referred to as pHIVenv (FIGS. 1 and 3).

The genomic HIV expression vectors pHIVluc and pHIVlucΔU3 are designedto transcribe HIV genomic RNA and subgenomic mRNAs and to express allHIV proteins except the envelope polyprotein (FIG. 1B). In thesevectors, a portion of the envelope gene has been deleted to accommodatea functional indicator gene cassette, in this case, “Firefly Luciferase”that is used to monitor the ability of the virus to replicate in thepresence or absence of anti-viral drugs. In pHIVlucΔU3, a portion of the3′ U3 region has been deleted to prevent transcription of viral RNAsfrom the 5′ LTR in infected cells.

Susceptibility assays for HIV-1 entry inhibitors were performed usingpackaging host cells consisting of the human embryonic kidney cell line293 (Cell Culture Facility, UC San Francisco, SF, Calif.) and targethost cells consisting of a human osteosarcoma (HOS) cell line expressingCD4 (HT4) plus CCR5, and CXCR4, or astrocytoma (U-87) cell linesexpressing either CD4 and CCR5 or CD4 and CXCR4.

Drug susceptibility testing was performed using pHIVenv and pHIVluc orpHIVlucΔU3. Pseudotyped HIV particles containing envelope proteinsencoded by the subject derived segment were produced by transfecting apackaging host cell (HEK 293) with resistance test vector DNA. Virusparticles were collected (˜48 h) after transfection and are used toinfect target cells (HT4/CCR5/CXCR4, or U-87/CD4/CXCR4, orU-87/CD4/CCR5) that express HIV receptors (i.e. CD4) and co-receptors(i.e. CXCR4, CCR5). After infection (˜72 h) the target cells are lysedand luciferase activity is measured. HIV must complete one round ofreplication to successfully infect the target host cell and produceluciferase activity. The amount of luciferase activity detected in theinfected cells is used as a direct measure of “infectivity” (FIGS. 1 and2). If for any reason (e.g. lack of the appropriate receptor orco-receptor, inhibitory drug activity, neutralizing antibody binding),the virus is unable to enter the target cell, luciferase activity isdiminished. Thus, the assay permits identification of co-receptor usageof the tested virus particles by measuring the ability of the particlesto enter cells expressing CD4 and either CCR5 or CXCR4 or both, in thepresence or absence of co-receptor inhibitors (FIGS. 3A and 3B).

Drug susceptibility is assessed by comparing the infectivity in theabsence of drug to infectivity in the presence of drug. Relative drugsusceptibility can be quantified by comparing the susceptibility of the“test” virus to the susceptibility of a well-characterized referencevirus (wildtype) derived from a molecular clone of HIV-1, for exampleNL4-3 or HXB2.

Packaging host cells were seeded in 10-cm-diameter dishes and weretransfected one day after plating with pHIVenv and pHIVluc orpHIVlucΔU3. Transfections were performed using a calcium-phosphateco-precipitation procedure. The cell culture media containing the DNAprecipitate was replaced with fresh medium, from one to 24 hours, aftertransfection. Cell culture media containing viral particles wastypically harvested 2 days after transfection and was passed through a0.45-mm filter. Before infection, target cells were plated in cellculture media. Entry inhibitor drugs were typically added to targetcells at the time of infection (one day prior to infection on occasion).Typically, 3 days after infection target cells were assayed forluciferase activity using the Steady-Glo reagent (Promega) and aluminometer.

7.2 Example 2

Identifying Envelope Amino Acid Substitutions/Mutations that AlterSusceptibility to Virus Entry Inhibitors

This example provides a means and method for identifying mutations inHIV-1 envelope that confer altered, e.g., reducedsusceptibility/resistance to virus entry inhibitors. This example alsoprovides a means and method for quantifying the degree of altered, e.g.,reduced susceptibility to entry inhibitors conferred by specificenvelope mutations.

Envelope sequences derived from subject samples, or individual clonesderived from subject samples, or envelope sequences engineered by sitedirected mutagenesis to contain specific mutations, were tested in theentry assay to quantify drug susceptibility based on awell-characterized reference standard (e.g. NL4-3, HXB2).

In one embodiment, susceptibility to longitudinal subject samples(viruses collected from the same subject at different timepoints) isevaluated. For example, susceptibility to entry inhibitors is measuredprior to initiating therapy, before or after changes in drug treatment,or before or after changes in virologic (RNA copy number), immunologic(CD4 T-cells), or clinical (opportunistic infection) markers of diseaseprogression.

7.2.1 Genotypic Analysis of Subject HIV Samples

Envelope sequences representing subject sample pools, or clones derivedfrom subject pools, can be analyzed by any broadly available DNAsequencing methods. In this example, subject HIV sample sequences weredetermined using viral RNA purification, RT/PCR and dideoxynucleotidechain terminator sequencing chemistry and capillary gel electrophoresis(Applied Biosystems, Foster City, Calif.). Envelope sequences of subjectvirus pools or clones were compared to reference sequences and othersubject samples. The genotypes of the viruses were examined forsequences that are different from the reference or pre-treatmentsequence and correlated to differences in entry inhibitorsusceptibility, as described below.

7.2.2 Entry Inhibitor Susceptibility of Site Directed Mutants

Genotypic changes that correlate with changes in fitness are evaluatedby constructing envelope expression vectors (pHIVenv) containing thespecific mutation on a defined, drug susceptible, genetic background(e.g. NL4-3 reference strain). Mutations may be incorporated aloneand/or in combination with other mutations that are thought to modulatethe entry inhibitor susceptibility. Envelope mutations are introducedinto pHIVenv vectors using any of the broadly available methods forsite-directed mutagenesis. In certain embodiments the mega-primer PCRmethod for site-directed mutagenesis is used (Sarkar, G. and Summer, S.S., 1990). A pHIVenv vector containing a specific envelope mutation orgroup of mutations are tested using the virus entry assay described inExample 1. Drug susceptibility of the virus containing envelopemutations is compared to the drug susceptibility of a geneticallydefined drug susceptible virus that lacks the specific mutations underevaluation. Observed changes in entry inhibitor susceptibility areattributed to the specific mutations introduced into the pHIVenv vector.

7.3 Example 3

Measuring Susceptibility to Virus Entry Inhibitors to Guide TreatmentDecisions

This example provides a means and method for using virus entry inhibitorsusceptibility to guide the treatment of HIV-1. This example furtherprovides a means and method for using virus entry inhibitorsusceptibility to guide the treatment of subjects that have receivedprevious antiretroviral treatment with a virus entry inhibitor. Thisinvention further provides a means and methods for using virus entryinhibitor susceptibility to guide the treatment of subjects that havenot received previous treatment with a virus entry inhibitor.

In one embodiment, the susceptibility of subject's viruses to virusentry inhibitors is used to guide the treatment of subjects failingantiretroviral regimens that include one or more virus entry inhibitors.Treatment failure (also referred to as virological failure) is generallydefined as partially suppressive antiviral treatment resulting indetectable levels of virus, which is typically measured in the subjectplasma. Guidance may include, but is not limited to, (a) clarificationof available drug treatment options, (b) selection of more activetreatment regimens, (c) clarification of the etiology of rising viralload in treated subjects (i.e. poor adherence, drug resistance), and (d)reduction in the use of inactive and potentially toxic drugs. In thisembodiment, resistance test vectors are derived from a subject virussamples and tested for susceptibility to various virus entry inhibitorsusing the phenotypic virus entry assay. Virus entry inhibitors mayinclude, but are not limited to, fusion inhibitors (e.g. T-20, T-1249),co-receptors antagonists (AMD3100, AMD8664, TAK779, PR0542, andpeperidin-lyl butane compounds) and CD4 antagonists (MAb B4).Appropriate treatment decisions are based on the results of the virusentry assay (e.g. see FIG. 4B) and additional relevant laboratory testresults and clinical information.

In another embodiment, the susceptibility of subject's viruses to virusentry inhibitors is used to guide the treatment of subjects that havenot been previously treated with antiretroviral regimens that includeone or more virus entry inhibitors. Guidance may include, but is notlimited to, (a) clarification of available drug treatment options, (b)selection of more active treatment regimens, (c) clarification of thebaseline susceptibility to virus entry inhibitors, and (d) reduction inthe use of inactive and potentially toxic drugs. Determining baselinesusceptibility of virus entry inhibitors in treatment naive subjects isimportant for two reasons. First, the natural susceptibility of virusesto entry inhibitors can vary widely (e.g. see FIG. 4A). Second, theincreased use of virus entry inhibitors will undoubtedly result in thegeneration of drug resistant variants that can be transmitted to newlyinfected individuals. In this embodiment, resistance test vectors arederived from a subject virus samples and tested for susceptibility tovarious virus entry inhibitors using the phenotypic virus entry assay.Virus entry inhibitors may include, but are not limited to, fusioninhibitors (e.g. T-20, T-1249), co-receptor antagonists (e.g. AMD3100,AMD8664, TAK-355, PR0542, and peperidin-lyl butane compounds) and CD4antagonists (MAb 5A8). Appropriate treatment decisions are based on theresults of the virus entry assay and additional relevant laboratory testresults and clinical information.

7.4 Example 4

Exploring the Effects of Co-Receptor Inhibitors on Homogeneous ViralPopulations

This example describes the results of experiments designed to assess theeffects of co-receptor inhibitors on homogeneous populations ofdual-tropic viruses. First, 35 gp160 envelope genes derived fromdifferent patient samples that were originally identified as dual-tropicwere cloned into an expression vector as described in Example 1. Tropismof pseudotyped virus was determined by measuring luciferase activity(relative light units, RLU) following infection of U87/CD4+CCR5+ orU87/CD4+CXCR4+ cells as described above. Inhibition of infection bysmall molecule CCR5-(Merck) and CXCR4-(AMD3100; AnorMED) inhibitors wasexamined on U87/CD4+CCR5+CXCR4+ cells, U87/CD4+CXCR4+ cells, andU87/CD4+CCR5+ cells. A subset of the envelope genes were also insertedinto an NL4-3 infectious clone and evaluated for their ability to infectPBMCs in the presence of the CCR5- or CXCR4-inhibitors.

Results from the first set of experiments showing tropism of the 35individual pseudotyped viruses are presented in Table 4, immediatelybelow. In Table 4, the individual clones have been sorted into clonesthat are primarily CCR5-tropic (shown as R5>>.times.4 in the table; alsoreferred to herein as Dual_R clones), clones that are dual tropic (shownas R5˜X4; also referred to as Dual clones) and clones that are primarilyCXCR4-tropic (shown as X4>>R5; also referred to as Dual_X clones).

TABLE 4 Normalized RLUs U87 U87 U87 Sample Dual Type CCR5+ CXCR4+CCR5+/CXCR4+ JRCSF R5 976,598 110 1,230,709 Clone 1 R5 >> X4 11,720 7628,691 Clone 2 R5 >> X4 572,726 2,563 459,781 Clone 3 R5 >> X4 627,6601,005 396,022 Clone 4 R5 >> X4 736,225 17,092 762,260 Clone 5 R5 >> X4885,752 14,650 693,807 Clone 6 R5 >> X4 961,696 199 535,129 Clone 7R5 >> X4 997,251 3,502 790,073 Clone 8 R5 >> X4 1,082,548 4,1941,023,988 Clone 9 R5 >> X4 1,082,628 2,328 1,075,651 Clone 10 R5 >> X41,096,117 11,969 1,106,647 Clone 11 R5 >> X4 1,116,961 1,317 821,801Clone 12 R5 >> X4 1,655,718 4,380 2,695,407 Clone 13 Clone 14 R5~X4203,669 177,006 241,521 Clone 15 R5~X4 5,552 6,471 19,785 Clone 16 R5~X436,172 20,792 23,688 Clone 17 R5~X4 43,106 97,388 57,455 Clone 18 R5~X444,826 134,845 94,003 Clone 19 R5~X4 75,845 31,952 41,526 Clone 20 R5~X4114,542 85,460 100,596 Clone 21 R5~X4 244,734 304,576 419,322 Clone 22R5~X4 247,316 117,635 182,245 Clone 23 R5~X4 369,291 345,867 414,392Clone 24 R5~X4 389,392 205,340 322,182 Clone 25 R5~X4 472,975 635,726843,164 Clone 26 R5~X4 707,184 435,623 499,922 Clone 27 R5~X4 827,294437,614 484,149 Clone 28 R5~X4 1,429,209 1,601,696 1,816,470 Clone 29Clone 30 X4 >> R5 256 9,210 11,700 Clone 31 X4 >> R5 824 71,954 87,239Clone 32 X4 >> R5 2,638 74,893 48,381 Clone 33 X4 >> R5 513 399,563468,71 Clone 34 X4 >> R5 28,427 655,790 433,996 Clone 35 X4 >> R5 11,153689,002 712,325 Clone 36 X4 >> R5 613 727,745 614,032 Clone 37 X4 >> R59,105 811,378 636,845 NL43 X4 65 602,816 724,602

Next, inhibition patterns for clones representing primarily CCR5-tropic,dual tropic, and primarily CXCR4-tropic viruses were determined. Inthese experiments, the ability of psuedotyped viruses to enter cellsexpressing CD4 and either or both of CCR5 and/or CXCR4 were tested inthe presence of the CCR5 inhibitor or the CXCR4 inhibitor. Results fromthis experiment are presented as FIG. 5.

As shown in FIG. 5, entry of primarily CCR5-tropic viruses intoCCR5-expressing and CCR5- and CXCR4-expressing cells was inhibited bythe CCR5-inhibitor. Entry of such viruses into cells expressing CCR5 andCXCR4 was not inhibited by the CXCR4-inhibitor. Similarly, entry ofprimarily CXCR4-tropic viruses into CXCR4-expressing and CCR5- andCXCR4-expressing cells was inhibited by the CXCR4 inhibitor, but entryof such viruses into cells expressing both CXCR4 and CCR5 was notcompletely inhibited by the CCR5-inhibitor. Entry of dual tropic virusesinto cells expressing only CXCR4 or CCR5 could be completely inhibitedby the appropriate co-receptor inhibitor, but neither could completelyinhibit entry of such viruses into cells expressing both co-receptors.Distribution of the percentage of inhibition for the individual dualtropic clones is shown as FIG. 6.

Taken together, these data indicate that entry of viruses that areprimarily CCR5 tropic into cells expressing both CXCR4 and CCR5 can beeffectively inhibited with CCR5 inhibitors, notwithstanding the viruses'weak ability to enter cells expressing CXCR4. Similarly, entry ofviruses that are primarily CXCR4-tropic into cells expressing both CXCR4and CCR5 can be effectively inhibited with CXCR4 inhibitors,notwithstanding the viruses' weak ability to enter cells expressingCXCR4.

To confirm that the entry phenotypes observed for the pseudotyped viralparticles conforms to the phenotypes of fully replication competentvirus, a subset of the envelope genes were inserted into an NL4-3infectious clone and evaluated for their ability to infect PBMCs in thepresence of the CCR5-inhibitor or AMD3100. Results from this experimentare presented in Table 5, below and presented graphically in FIG. 7.

TABLE 5 The CCR5− CCR5− inhi- inhi- bitor AMD bitor/AMD Clone Tropism %inhi- % inhi- % inhi- ID Subtype bition bition bition JRCSF R5_JRCSF R5100%  11% 100% Clone 4 R5 >> X4_1 R5 >> X4 99% −1% 100% Clone 11 R5 >>X4_2 R5 >> X4 100%  −3% 100% Clone 26 R5~X4_1 R5~X4 43% 41% 100% Clone25 R5~X4_2 R5~X4 26% 27% 100% Clone 28 R5~X4_3 R5~X4 32% 19% 100% Clone22 R5~X4_4 R5~X4 53% −12%   99% Clone 34 X4 >> R5_1 X4 >> R5 14% 99% 99% Clone 33 X4 >> R5_2 X4 >> R5 17% 99%  98% Clone 31 X4 >> R5_3 X4 >>R5 48% 98%  99% NL43 X4_NL43 X4 29% 99% 100%

As shown in FIG. 7 and Table 5, the results observed for the fullyreplication competent viruses were consistent with those obtained frompseudotyped viral particles: entry of Dual_R-tropic viruses could besubstantially inhibited by the CCR5 inhibitor, while entry ofDual_X-tropic viruses could be substantially inhibited by the CXCR4inhibitor. Entry of the dual-tropic viruses could be partially inhibitedwith either the CCR5-inhibitor or the CXCR4-inhibitor, but could not becompletely inhibited.

Finally, it was observed that individual patients were infected withclones of differing subtypes; for example, one subject (from whichclones 9, 36, and 37 were isolated) was infected with one primarilyCCR5-tropic viral subpopulation and at least one primarily CXCR4-tropicviral subpopulation (Table 4). Accordingly, the relative contributionsof different subpopulations to entry was assessed in another set ofexperiments as described below.

7.5 Example 5

Analysis of Suppression of Primarily CXCR4-Tropic Viruses inHeterogeneous Populations Infecting Patients

This example describes the results of experiments performed to assesssuppression of dual-tropic HIV-1 variants by the CXCR4 inhibitor AMD3100patients over time. In brief, the results of these experiments indicatethat such suppression is associated with efficiency of CXCR4 use andclonal composition of the baseline virus population.

First, samples from 26 subjects administered AMD3100 for 10 days wereobtained prior to therapy and on day 11 following the 10 days oftherapy. Co-receptor tropism of the viral populations from both sets ofsamples were then determined according to Example 1. The results of thepopulation analysis were originally published in Hendrix et al., 2004, JAcquir Immune Defic Syndr. 37(2):1253-62.

To further characterize these populations, individual clones from thepatients identified as infected with dual-tropic viral populations weresubjected to further analysis as described hereinafter. First, patientswere classified by the response of the patient virus to the CXCR4inhibitor into patients that experienced suppression of CXCR4-tropicviruses as a relative proportion of the population (Suppressors) andpatients that did not experience suppression of CXCR4-tropic viruses(Non-suppressors). These classifications are shown in FIG. 8.

To begin to assess viral populations of the patients that experiencedsuppression versus the patients that did not, the tropism phenotypes of20-50 individual functional envelope clones from each of the patientsidentified as DM (dual/mixed)-tropic were determined according toExample 1, and the percentage of CCR5-tropic, CXCR4-tropic, ordual-tropic clones before and after therapy were determined. Resultsfrom this analysis are presented in FIG. 9.

These results indicate that suppression of CXCR4-tropic virus dependedon the viral composition at baseline. Populations comprising 100% dualviruses, e.g., a homogeneous population of dual-tropic viruses, were notsuppressed by 10 days of AMD3100 therapy, while populations containingmixtures of CCR5-tropic and CXCR4-tropic or dual-tropic virusesexperienced suppression. That is, following treatment with AMD3100, insubjects with mixed populations, entry into cells using the CXCR4co-receptor was efficiently blocked while entry using the CCR5co-receptor was unaffected. This led to substantial shifts of tropismaway from CXCR4-tropic viruses and towards CCR5-tropic viruses in thesesubjects.

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What is claimed is:
 1. A method for determining whether a subjectinfected with a dual-mixed tropic population of HIV would benefit fromCXCR4-inhibitor therapy, comprising determining whether the HIVpopulation is a homogeneous or heterogeneous population of HIV bydetermining tropism for at least 20 HIV from the population, and a.determining that the subject would benefit from CXCR4-inhibitor therapyif the subject is infected with a homogeneous population of HIV and thehomogeneous population is Dual_X tropic, wherein the HIV is Dual_Xtropic if the HIV has at least 10-fold greater ability to enter cellsexpressing the CXCR4 co-receptor relative to the HIV's ability to entercells co-expressing CCR5; and treating the subject with an effectiveamount of a CXCR4 inhibitor if the homogeneous population is Dual_Xtropic; or b. determining that the subject would benefit fromCXCR4-inhibitor therapy if the subject is infected with a heterogeneouspopulation of HIV and the heterogeneous population of HIV comprisessubstantially more CXCR4-tropic or Dual_X tropic HIV than totalCCR5-tropic, Dual_R tropic, and dual-tropic HIV, wherein the HIV isDual_X tropic if the HIV has at least 10-fold greater ability to entercells expressing the CXCR4 co-receptor relative to the HIV's ability toenter cells co-expressing CCR5, and wherein the HIV is Dual_R tropic ifthe HIV has at least 10-fold greater ability to enter cells expressingthe CCR5 co-receptor relative to the HIV's ability to enter cellsco-expressing CXCR4; and treating the subject with an effective amountof a CXCR4 inhibitor if the heterogeneous population of HIV comprisessubstantially more CXCR4-tropic or Dual_X tropic HIV than totalCCR5-tropic, Dual_R tropic, and dual-tropic HIV.
 2. The method of claim1, wherein the HIV population is homogeneous.
 3. The method of claim 1,wherein the HIV population is heterogeneous.
 4. The method of claim 1,wherein the step of determining whether the HIV population is ahomogeneous or heterogeneous population is performed by determiningtropism for at least 100 HIV infecting the subject.
 5. A method fordetermining whether a subject infected with a dual-mixed tropicpopulation of HIV would benefit from CCR5-inhibitor therapy, comprisingdetermining whether the HIV population is a homogeneous or heterogeneouspopulation of HIV by determining tropism for at least 20 HIV from thepopulation, and a. determining that the subject would benefit fromCCR5-inhibitor therapy if the subject is infected with a homogeneouspopulation of HIV and the homogeneous population is Dual_R tropic,wherein the HIV is Dual_R tropic if the HIV has at least 10-fold greaterability to enter cells expressing the CCR5 co-receptor relative to theHIV's ability to enter cells co-expressing CXCR4; and treating thesubject with an effective amount of a CCR5 inhibitor if the homogeneouspopulation is Dual_R tropic; or b. determining that the subject wouldbenefit from CCR5-inhibitor therapy if the subject is infected with aheterogeneous population of HIV and the heterogeneous population of HIVcomprises substantially more CCR5-tropic or Dual_R tropic HIV than totalCXCR4-tropic, Dual_X tropic, and dual-tropic HIV, wherein the HIV isDual_R tropic if the HIV has at least 10-fold greater ability to entercells expressing the CCR5 co-receptor relative to the HIV's ability toenter cells co-expressing CXCR4, and wherein the HIV is Dual_X tropic ifthe HIV has at least 10-fold greater ability to enter cells expressingthe CXCR4 co-receptor relative to the HIV's ability to enter cellsco-expressing CCR5; and treating the subject with an effective amount ofa CCR5 inhibitor if the heterogeneous population of HIV comprisessubstantially more CCR5-tropic or Dual_R tropic HIV than totalCXCR4-tropic, Dual_X tropic, and dual-tropic HIV.
 6. The method of claim5, wherein the HIV population is homogeneous.
 7. The method of claim 5,wherein the HIV population is heterogeneous.
 8. The method of claim 5,wherein the step of determining whether the HIV population is ahomogeneous or heterogeneous population is performed by determiningtropism for at least 100 HIV infecting the subject.
 9. The method ofclaim 1, wherein the step of determining whether the HIV population is ahomogeneous or heterogeneous population is performed by determiningtropism for at least 112 HIV from the population.
 10. The method ofclaim 1, wherein the step of determining whether the HIV population is ahomogeneous or heterogeneous population is performed by determiningtropism for at least 125 HIV from the population.
 11. The method ofclaim 1, wherein the step of determining whether the HIV population is ahomogeneous or heterogeneous population is performed by determiningtropism for at least 137 HIV from the population.
 12. The method ofclaim 1, wherein the step of determining whether the HIV population is ahomogeneous or heterogeneous population is performed by determiningtropism for at least 150 HIV from the population.
 13. The method ofclaim 1, wherein the step of determining whether the HIV population is ahomogeneous or heterogeneous population is performed by determiningtropism for at least 175 HIV from the population.
 14. The method ofclaim 1, wherein the step of determining whether the HIV population is ahomogeneous or heterogeneous population is performed by determiningtropism for at least 200 HIV from the population.
 15. The method ofclaim 5, wherein the step of determining whether the HIV population is ahomogeneous or heterogeneous population is performed by determiningtropism for at least 112 HIV from the population.
 16. The method ofclaim 5, wherein the step of determining whether the HIV population is ahomogeneous or heterogeneous population is performed by determiningtropism for at least 125 HIV from the population.
 17. The method ofclaim 5, wherein the step of determining whether the HIV population is ahomogeneous or heterogeneous population is performed by determiningtropism for at least 137 HIV from the population.
 18. The method ofclaim 5, wherein the step of determining whether the HIV population is ahomogeneous or heterogeneous population is performed by determiningtropism for at least 150 HIV from the population.
 19. The method ofclaim 5, wherein the step of determining whether the HIV population is ahomogeneous or heterogeneous population is performed by determiningtropism for at least 175 HIV from the population.
 20. The method ofclaim 5, wherein the step of determining whether the HIV population is ahomogeneous or heterogeneous population is performed by determiningtropism for at least 200 HIV from the population.